The attenuated PfSPZ vaccine being developed by Sanaria is designed to evoke a robust adaptive immune response that will prevent mosquito transmitted sporozoites from establishing clinical malaria in vaccinated individuals. Based on decades of studies with live and attenuated sporozoites, it is expected that such a successful immune response will involve both humoral and cell mediated mechanisms where the latter component is critically dependent on activated CD8+ T cells that are primed by the vaccine and are subsequently able to destroy even the rare infected liver cell. We propose to develop assays that will robustly detect the presence of anti-P. falciparum sporozoite and liver stage CTLs with a high degree of sensitivity. Antigen-specific T cells against subunit vaccines are routinely detected by indirect measures of cytokine secretion using established assays that assess immune responses against defined antigens or epitopes from these antigens. But Sanaria's attenuated whole-organism PfSPZ vaccine is likely to elicit T cell responses against a multitude of antigens limited only by the size of the parasite genome. Moreover, the identity of the relevant immunogenic and protective epitopes within the genomic repertoire is currently unknown. Therefore, an assay that is unbiased to specific peptides and that directly tests the functional activity of T cells against infected target cells, in this case, a hepatocyte is more meaningful in the long run and can eventually be more robustly predictive of vaccine efficacy. Towards this goal we will develop a modified Inhibition of Liver-Stage Development Assay (ILSDA) with four specific aims 1) Increase robustness of ILSDA 2) Establish the capacity to demonstrate hepatocyte killing using specific CTL clones 3) Adapt the improved ILSDA to a suitable high-throughput platform and 4) Validate the improved high throughput ILSDA using PBMCs from immunized individuals. In its final version we expect to demonstrate T cell subset dependence of the activity by depleting specific T cell subsets, the MHC restriction by incubating with antibodies to specific HLA molecules, and the antigen specificity by infecting with P. vivax sporozoites. This functional assay which measures T cell activity against whole parasites is critical for the development of Sanaria's PfSPZ Vaccine, and could play an equally important role for other pre-erythrocytic stage malaria vaccines designed to induce protective T cell responses. PUBLIC HEALTH RELEVANCE: This proposal aims to develop a robust high throughput assay that can detect highly specific cytotoxic T cells primed by Sanaria's PfSPZ vaccine or any other pre-erythrocytic malaria vaccine. Building on the essential Inhibition of Liver stage Development Assay (ILSDA) that was originally described by Sanaria's Chief Scientific Officer and co-investigator on this grant, Dr. Stephen Hoffman, the hepatocyte cytotoxicity assay will use the biological inhibition of parasite growth within infected liver cells as a direct and reliable measure of the functional activity of primed lymphocytes in immunized individuals.